The experiments in this proposal are designed to address important outstanding questions in photoreceptor physiology, and to use the most powerful methods presently available to answer these questions. In collaboration with molecular biologists, this project will use animals with targeted mutations in essential photoreceptor proteins in order to probe basic features of photoreceptor function, in direct response to the program objective of the Retinal Disease Program of the NEI to analyze the mechanisms underlying light adaptation and recovery following phototransduction. These experiments will take advantage of techniques recently developed in the PI's laboratory that permit the fast perfusion of the small outer segments of mouse rods, and the bleaching and regeneration of pigment in isolated cells. One of the principal goals of this proposal is to ask how rod responses recover after stimulation with light, and what causes this recovery to be accelerated as the intensity of background illumination is increased, so that we become better able to detect change and motion in bright light. Is the rate of photoreceptor recovery modulated by Ca2+, and if so, by what process? Similar approaches will be used to study mechanisms of photoreceptor light adaptation. Recent experiments have shown that adaptation cannot be completely explained by the usually proposed mechanisms of regulation of guanylyl cyclase, rhodopsin phosphorylation, and channel opening, but that a newly discovered component probably produced by modulation of phosphodiesterase also makes an important contribution. Is light adaptation in mammals controlled by outer segment Ca2+ concentration? If so, does Ca2+ regulate the rate of phosphodiesterase decay as well as the rate of cGMP synthesis? Does the desensitization produced by bright bleaching light also have a phosphodiesterase component, and is bleaching desensitization produced by a mechanism essentially identical to the one producing desensitization in maintained background light? If these questions can be answered, they will move photoreceptor transduction in a new direction and stimulate additional research into the biochemistry of vision. Taken together, the experiments of this proposal will contribute to a more detailed understanding of the physiology of G-protein cascades throughout the body. PUBLIC HEALTH RELEVANCE: The great majority of diseases of the retina are caused by disorder or degeneration of the photoreceptors, the cells in the eye that convert light into an electrical signal. This proposal seeks to understand basic mechanisms of photoreceptor function, particularly adaptation to maintained illumination and recovery after exposure to light, which are known to be implicated in genetically inherited retinal diseases including night blindness, bradyopsin, and Leber's amaurosis. This study will support an important program objective of the National Eye Institute of the NIH to analyze the mechanisms underlying light adaptation and recovery following phototransduction, and this work will also contribute to a more detailed understanding of the physiology of signal transduction throughout the body.